U.S. patent application number 11/445486 was filed with the patent office on 2007-01-04 for test system.
Invention is credited to Paul Chambers, Wah On Ho, John J. Rippeth.
Application Number | 20070000777 11/445486 |
Document ID | / |
Family ID | 37588177 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070000777 |
Kind Code |
A1 |
Ho; Wah On ; et al. |
January 4, 2007 |
Test system
Abstract
A test system for measuring analyte concentration in a fluid
sample includes: (i) a capillary-fill biosensor (20) having a
working electrode (24), a reference electrode (22) and a separate
counter electrode (23), arranged such that a fluid sample which
flows evenly along the capillary flow path will substantially
completely cover the reference electrode (22) before the fluid
sample makes contact with any part of the counter electrode (23);
and (ii) a test meter (42) for receiving the biosensor (20), the
meter (42) including: first signal circuitry (31) for producing a
first signal when an electrical circuit is detected between the
reference electrode (22) and the working electrode (24); second
signal circuitry (33) for producing a second signal when an
electrical circuit is detected between the counter electrode (23)
and at least one of the reference electrode (22) and the working
electrode (24); a timer (35) for determining the time interval
between production of said first signal and said second signal; and
a processor (32) for triggering an error condition if said time
interval exceeds a preset value or if said second signal is not
produced within a preset time after said first signal is
produced.
Inventors: |
Ho; Wah On; (Colchester,
GB) ; Rippeth; John J.; (Ipswich, GB) ;
Chambers; Paul; (Ipswich, GB) |
Correspondence
Address: |
O'KEEFE, EGAN AND PETERMAN, L.L.P.;Building C
Suite 200
1101 Capital of Texas Highway South
Austin
TX
78746
US
|
Family ID: |
37588177 |
Appl. No.: |
11/445486 |
Filed: |
June 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60687712 |
Jun 6, 2005 |
|
|
|
Current U.S.
Class: |
204/403.14 ;
204/406 |
Current CPC
Class: |
C12Q 1/001 20130101 |
Class at
Publication: |
204/403.14 ;
204/406 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00; G01N 27/26 20060101 G01N027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2005 |
GB |
GB0511270.1 |
Claims
1. A test system comprising: (i) a capillary-fill biosensor for
indicating electrochemically the catalytic activity of an enzyme in
the presence of a fluid sample containing an analyte acted upon by
said enzyme; the biosensor having a fluid inlet, a fluid outlet,
and a capillary flow path connecting the fluid inlet and the fluid
outlet; the biosensor further including a working electrode, a
reference electrode and a counter electrode located within the
capillary flow path; the working electrode including a
catalytically-active quantity of said enzyme; wherein the reference
electrode is separate from the counter electrode, and the reference
and counter electrodes are arranged such that a fluid sample which
flows evenly along the capillary flow path from said fluid inlet
will substantially completely cover the reference electrode before
the fluid sample makes contact with any part of the counter
electrode; and (ii) a test meter for receiving said biosensor, the
meter comprising: contacts for making electrical contact with each
electrode on said biosensor; circuitry for performing an
electrochemical measurement on said biosensor to produce a measured
electrochemical value; circuitry for determining an analyte
concentration value for a specified biological fluid from said
measured electrochemical value; and circuitry for indicating a
determined analyte concentration value; the meter further
comprising: first signal circuitry for producing a first signal
when an electrical circuit is detected between the reference
electrode and the working electrode; second signal circuitry for
producing a second signal when an electrical circuit is detected
between the counter electrode and at least one of the reference
electrode and the working electrode; a timer for determining the
time interval between production of said first signal and said
second signal; and a processor for triggering an error condition if
said time interval exceeds a preset value or if said second signal
is not produced within a preset time after said first signal is
produced.
2. A system according to claim 1, further comprising: a switch for
releasably connecting the reference electrode and the counter
electrode, the switch being closed before an electrical circuit has
been detected between the reference electrode and the working
electrode; and circuitry for opening the switch when an electrical
circuit has been detected between the reference electrode and the
working electrode.
3. A system according to claim 2, wherein said circuitry for
opening the switch is a potentiostat circuit which makes a feedback
loop when an electrical circuit has been made between the working
electrode and the counter electrode.
4. A system according to claim 2, wherein the reference electrode
will go open circuit when the switch is opened.
5. A system according to claim 1, wherein the biosensor comprises:
a first substrate; a second substrate overlying at least a part of
the first substrate; the working electrode, the counter electrode,
and the reference electrode each being provided on either one of
the substrates; conductive tracks connected to said working,
counter and reference electrodes for making electrical connections
with a test meter apparatus; a spacer layer having a channel
therein and disposed between the first substrate and the second
substrate, the spacer layer channel co-operating with adjacent
surfaces to define the capillary flow path which extends from an
edge of at least one of said substrates to said electrodes.
6. A system according to claim 1, wherein each of said electrodes
is non-mediated and includes a base layer comprising particles of a
platinum-group metal or platinum-group metal oxide bonded together
by a resin.
7. A system according to claim 6, wherein each of said electrodes
further includes a top layer comprising a buffer on the base layer,
and a catalytically-active quantity of an oxidoreductase enzyme in
at least one of the top layer and the base layer.
8. A system according to claim 1, wherein the counter electrode is
downstream of the working electrode in the capillary flow path.
9. A system according claim 1, wherein the working electrode is
positioned such that a fluid sample which flows evenly along the
capillary flow path from said edge will make contact with the
reference electrode no later than it will make contact with the
working electrode.
10. A test system comprising: (i) a capillary-fill biosensor having
a working electrode, a reference electrode and a counter electrode
located within a capillary flow path between a fluid inlet and a
fluid outlet; wherein the reference electrode is separate from the
counter electrode, and the reference and counter electrodes are
arranged such that a fluid sample which flows evenly along the
capillary flow path from said fluid inlet will substantially
completely cover the reference electrode before the fluid sample
makes contact with any part of the counter electrode; and (ii) a
test meter for receiving said biosensor, said test meter including:
circuitry for producing a first signal when an electrical circuit
is detected between the reference electrode and the working
electrode; circuitry for producing a second signal when an
electrical circuit is detected between the counter electrode and at
least one of the reference electrode and the working electrode; a
timer for determining the time interval between production of said
first signal and said second signal; and a processor for triggering
an error condition if said time interval exceeds a preset value or
if said second signal is not produced within a preset time after
said first signal is produced.
11. A test system comprising: (i) a capillary-fill biosensor for
indicating electrochemically the catalytic activity of an enzyme in
the presence of a fluid sample containing an analyte acted upon by
said enzyme; the biosensor having a working electrode, a reference
electrode and a counter electrode located within a capillary flow
path between a fluid inlet and a fluid outlet; the working
electrode including a catalytically-active quantity of said enzyme;
wherein the reference electrode is separate from the counter
electrode, and the reference and counter electrodes are arranged
such that a fluid sample which flows evenly along the capillary
flow path from said fluid inlet will substantially completely cover
the reference electrode before the fluid sample makes contact with
any part of the counter electrode; and (ii) a test meter for
receiving said biosensor, the meter comprising: means for making
electrical contact with each electrode on said biosensor; circuitry
for performing an electrochemical measurement on said biosensor to
produce a measured electrochemical value; means for determining an
analyte concentration value for a specified biological fluid from
said measured electrochemical value; and means for indicating a
determined analyte concentration value; the meter further
comprising: first signal means for producing a first signal when an
electrical circuit is detected between the reference electrode and
the working electrode; second signal means for producing a second
signal when an electrical circuit is detected between the counter
electrode and at least one of the reference electrode and the
working electrode; timing means for determining the time interval
between production of said first signal and said second signal; and
processing means for triggering an error condition if said time
interval exceeds a preset value or if said second signal is not
produced within a preset time after said first signal is
produced.
12. A test meter for use with a biosensor, the meter comprising: a
first electrode contact for making an electrical connection with a
working electrode on the biosensor; a second electrode contact for
making electrical contact with a reference electrode on the
biosensor; a third electrode contact for making an electrical
connection with a counter electrode on the biosensor; a processor
for receiving an input signal from said electrode contacts; a data
store containing data relating to a specified biological fluid and
accessible by said processor; circuitry for measuring an electrical
property of a fluid sample applied to a biosensor when the
biosensor is inserted in the meter; wherein the processor is
arranged in operation to access the data store and to output an
output signal representative of analyte concentration derived from
the input signal and data contained in the accessed data store; an
output device for visually or audibly outputting information
dependent on said output signal; first signal circuitry for
producing a first signal when an electrical circuit is detected
between said first electrode contact and said second electrode
contact; second signal circuitry for producing a second signal when
an electrical circuit is detected between said third electrode
contact and at least one of said first electrode contact and said
second electrode contact; a timer for determining the time interval
between production of said first signal and said second signal; and
circuitry for triggering an error condition if said time interval
exceeds a preset value or if said second signal is not produced
within a preset time after said first signal is produced.
13. A test meter according to claim 12, further comprising: a
switch for releasably connecting the second electrode contact and
the third electrode contact, the switch being closed before an
electrical circuit has been detected; and switch-opening circuitry
for opening the switch when an electrical circuit has been
detected.
14. A test meter according to claim 13, wherein said switch-opening
circuitry is a potentiostat circuit which makes a feedback loop
when an electrical circuit has been made between the first
electrode contact and the third electrode contact.
15. A test meter according to claim 13, wherein the second
electrode contact will go open circuit when the switch is
opened.
16. A test meter for use with a biosensor, the meter comprising: a
first electrode contact for making an electrical connection with a
working electrode on the biosensor; a second electrode contact for
making electrical contact with a reference electrode on the
biosensor; a third electrode contact for making an electrical
connection with a counter electrode on the biosensor; a processor
for receiving an input signal from said electrode contacts; storage
means containing data relating to a specified biological fluid and
accessible by said processor; means for measuring an electrical
property of a fluid sample applied to a biosensor when the
biosensor is inserted in the meter; wherein the processor is
arranged in operation to access the storage means and to output an
output signal representative of analyte concentration derived from
the input signal and data contained in the accessed storage means;
means for visually or audibly outputting information dependent on
said output signal; a switch for releasably connecting the second
electrode contact and the third electrode contact, the switch being
closed before a first electrical circuit has been detected;
potentiostat circuitry for opening the switch when said first
electrical circuit has been detected, which will cause the second
electrode contact to go open circuit; said circuitry being adapted
to make a feedback loop when a second electrical circuit has been
made between the first electrode contact and the third electrode
contact; timing means for determining the time interval between
detection of said first electrical circuit and detection of said
second electrical circuit; and means for triggering an error
condition if said time interval exceeds a preset value or if said
second electrical circuit is not detected within a preset time
after said first electrical circuit.
Description
[0001] This application claims priority to co-pending U.S.
provisional application Ser. No. 60/687,712 filed on Jun. 6.sup.th
2005, which is entitled "Test System" the disclosure of which is
incorporated herein by reference. This application claims priority
to co-pending United Kingdom patent application serial number
0511270.1 filed on Jun. 3.sup.rd 2005, which is entitled "Test
System" the disclosure of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a test system for measuring
analyte concentration in a fluid sample. The invention also
provides a biosensor for use in the system, notably a biosensor for
measuring analyte concentration in biological fluids, for example
glucose in whole blood.
[0004] 2. Description of the Prior Art
[0005] Biosensors typically include an enzyme electrode comprising
an enzyme layered on or mixed with an electrically conductive
substrate, for example a non-mediated enzyme electrode such as
described in US 2004/0061841. The electrodes respond
electrochemically to the catalytic activity of the enzyme in the
presence of a suitable analyte.
[0006] Electrochemical biosensors are well known in the art. They
are used in measurement techniques including amperometry,
coulometry and potentiometry. The biosensor comprises a working
electrode and a counter electrode to complete an electric circuit.
A reference electrode may also be used, to help maintain a constant
potential between the working and counter electrodes. The reference
and counter electrodes may be combined as a reference/counter
electrode.
[0007] Typically the enzyme is an oxidoreductase, for example
glucose oxidase, cholesterol oxidase, or lactate oxidase, which
produces hydrogen peroxide according to the reaction:
analyte+O.sub.2--[oxidase].fwdarw.oxidised
product+H.sub.2O.sub.2.
[0008] In an amperometric measurement, the peroxide is oxidised at
a fixed-potential working electrode as follows:
H.sub.2O.sub.2.fwdarw.O.sub.2+2H.sup.++2e.sup.-.
[0009] Electrochemical oxidation of hydrogen peroxide at platinum
centres on the working electrode results in transfer of electrons
from the peroxide to the electrode producing a current which is
proportional to the analyte concentration. Where glucose is the
analyte, the oxidised product is gluconolactone.
[0010] In coulometric measurement, the current passed during
completion or near completion of electrolysis of the analyte is
measured and integrated to give a value of charge passed. The
charge passed is related to the quantity of analyte present in a
sample so that if the sample volume is known the analyte
concentration can be determined. In potentiometric measurement, a
potential generated by the reaction is measured at one or more
points in time and related to the initial analyte concentration.
The various electrochemical measurement techniques are well known
to those skilled in the art.
[0011] Typically, electrochemical measurement begins automatically
when the fluid sample completes an electrical circuit between the
working and counter electrodes. Getting an accurate reading can be
a problem when a blood sample incompletely covers the working
electrode because the amount of current or measured charge is less
than when the working electrode is fully covered. If a user
attempts to top-up the sample by applying a second drop of blood
(`double-dosing`) this has the effect of reducing the precision of
the measurement and increasing the response as the addition of
extra blood causes a non-faradaic charging peak to occur when more
of the electrode area is covered by the second sample.
[0012] It has been proposed to reduce the problem of incomplete
fill by employing a pair of fill-detection electrodes in the fluid
path, with the working and counter electrodes inbetween. A
measurement is only taken when a circuit has been completed between
the fill electrodes. However, this arrangement adds complexity to
the system and does not address the problems of double-dosing by
the user.
SUMMARY OF THE INVENTION
[0013] According to an aspect of the present invention there is
provided a test system comprising: [0014] (i) a capillary-fill
biosensor having a working electrode, a reference electrode and a
counter electrode located within a capillary flow path between a
fluid inlet and a fluid outlet; [0015] wherein the reference
electrode is separate from the counter electrode, and the reference
and counter electrodes are arranged such that a fluid sample which
flows evenly along the capillary flow path from said fluid inlet
will substantially completely cover the reference electrode before
the fluid sample makes contact with any part of the counter
electrode; and [0016] (ii) a test meter including: [0017] circuitry
for producing a first signal when an electrical circuit is detected
between the reference electrode and the working electrode; [0018]
circuitry for producing a second signal when an electrical circuit
is detected between the counter electrode and at least one of the
reference electrode and the working electrode; [0019] a timer for
determining the time interval between production of said first
signal and said second signal; and [0020] a processor for
triggering an error condition if said time interval exceeds a
preset value or if said second signal is not produced within a
preset time after said first signal is produced.
[0021] The reference electrode provides a stable reference point
against which the voltage of the working electrode may be measured,
and enables a desired potential to be maintained between the
working and counter electrodes. The system is more stable than two
electrode systems with a combined counter and reference electrode.
In a preferred embodiment, all three electrodes are formed from the
same materials, and are preferably non-mediated electrodes. In a
particularly preferred embodiment, each electrode comprises an
electrically-conductive base layer comprising particles of a
platinum-group metal or platinum-group metal oxide bonded together
by a resin, a top layer comprising a buffer on the base layer, and
a catalytically-active quantity of an oxidoreductase enzyme in at
least one of the top layer and the base layer. The electrodes may
be manufactured as described for the working electrode in US
2004/0061841, the contents of which are incorporated herein by
reference. Manufacturing all three electrodes from the same
materials and arrangement of layers simplifies the manufacturing
process. We have found that the preferred system is also more
sensitive and gives a more linear response than one employing
electrodes which are a combination of platinised carbon and
silver/silver chloride.
[0022] In a preferred embodiment, the working electrode is
positioned such that a fluid sample which flows evenly along the
capillary flow path from the edge will make contact with the
reference electrode no later than it will make contact with the
working electrode. Completion of a circuit by the fluid sample
between the reference and working electrodes will signal that the
fluid sample has made contact with the working electrode. Moreover,
the absence of this signal will indicate that the fluid sample has
not made contact with the working electrode. Under this
circumstance, double dosing may be carried out without a circuit
having been established between the working and reference
electrodes.
[0023] The counter electrode may be located sufficiently downstream
of the reference electrode to ensure that, when the counter
electrode completes a circuit with the working electrode, at least
a substantial portion of the working electrode will be in contact
with the fluid sample. In a preferred embodiment, the counter
electrode is located such that a fluid sample which flows evenly
along the capillary path from the edge will substantially
completely cover the working electrode before the fluid sample
makes contact with any part of the counter electrode. A timer may
be initiated when a first electrical circuit is completed between
the reference electrode and the working electrode. If electrical
contact is not made between the working electrode and the counter
electrode within a specified period of time from completion of the
first circuit, the meter may signal that the biosensor has received
insufficient sample. Thus, the system may provide both short-fill
and insufficient-fill detection.
[0024] Each electrode may be provided on either substrate, although
it is preferred that all electrodes are provided on a single
substrate for ease of manufacture.
[0025] The error condition may comprise a visible and/or audible
warning, for example a display message warning that insufficient
blood has been applied to a biosensor, optionally with an
instruction to remove the biosensor from the meter and start again
with a fresh biosensor. The error condition may also prevent the
output of an analyte concentration value, thus preventing a reading
being taken by double dosing.
[0026] It will be understood that the processor, the storage means,
the signal means, the timing means, and the means for measuring the
electrical property may each be provided as separate components or
that any or all of them may provided in combination, for example in
a single processing unit.
[0027] In a preferred embodiment, the meter has circuitry for
releasably establishing an electrical connection between the
reference electrode and the counter electrode. The reference
electrode may therefore initially function as a combined
reference/counter electrode. When a current is detected between the
reference electrode and the working electrode, indicating entry of
a fluid sample, the first signal causes the processor to break the
electrical connection. The reference electrode then goes open
circuit and no current flows. When the fluid sample reaches the
working electrode, a circuit is created and the second signal is
triggered.
[0028] Other aspects and benefits of the invention will appear in
the following specification, drawings and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The invention will now be further described, by way of
example, with reference to the following drawings in which:
[0030] FIGS. 1 and 2 show stages in the formation of a biosensor in
accordance with aspects of the present invention;
[0031] FIGS. 3 and 4 are graphs showing current responses of the
biosensors of, respectively, FIGS. 1 and 2 for samples of venous
blood having different glucose concentrations;
[0032] FIG. 5 is a schematic representation of a meter for use with
the biosensors of FIGS. 1 and 2; and
[0033] FIG. 6 is a circuit diagram of a potentiostat circuit for
the meter of FIG. 5.
DETAILED DESCRIPTION
[0034] When used herein, the following definitions define the
stated term:
[0035] "Amperometry" includes steady-state amperometry,
chronoamperometry, and Cottrell-type measurements.
[0036] A "biological fluid" is any body fluid in which the analyte
can be measured. Examples include blood, sweat, urine, interstitial
fluid, dermal fluid, and tears.
[0037] A "biosensor" is a device for detecting the presence or
concentration of an analyte in a biological fluid by means of
electrochemical oxidation and reduction reactions transduced to an
electrical signal that can be correlated to the presence or
concentration of analyte.
[0038] "Blood" includes whole blood and fluid components of whole
blood, for example plasma and serum.
[0039] "Coulometry" is the determination of charge passed or
projected to pass during complete or near-complete electrolysis of
the analyte. The determination may be made using a single
measurement or multiple measurements of a decaying current and
elapsed time during electrolysis of a sample.
[0040] A "counter electrode" is one or more electrodes paired with
the working electrode, through which passes a current equal in
magnitude and opposite in sign to the current passed through the
working electrode.
[0041] "Electrolysis" is the electrooxidation or electroreduction
of a compound either directly at an electrode or via one or more
mediators.
[0042] A "faradaic current" is a current corresponding to the
reduction or oxidation of a chemical substance. The net faradaic
current is the algebraic sum of all the faradaic currents flowing
through a working electrode.
[0043] A "mediated biosensor" is a biosensor which includes a
significant quantity of a mediator.
[0044] A "mediator" is an electron carrier which, in an oxidised
form, accepts electrons from an enzyme and then, in a reduced
state, transports the electrons to an electrode where it becomes
re-oxidised. Examples of mediators include ferrocene, ferrocene
derivatives, ferricyanide, osmium complexes,
2,6-dichlorophenolindophenol, Nile Blue, and Medola Blue; see, for
example: U.S. Pat. No. 5,708,247, U.S. Pat. No. 6,241,862, U.S.
Pat. No. 6,436,256, WO 98/55856, and WO 99/13100.
[0045] A "non-mediated biosensor" is a biosensor which does not
include a significant quantity of a mediator.
[0046] "Potentiometry" is the measurement of electrical potential
under conditions of low or no current flow, which may be used to
determine the presence or quantity of analyte in a fluid.
[0047] A "reference electrode" is an electrode that has a
substantially stable equilibrium electrode potential. It can be
used as a reference point against which the potential of other
electrodes, notably the working electrode, can be measured.
[0048] A "working electrode" is an electrode at which analyte
undergoes electrolysis.
Preparation of BSA-Pt/Carbon
[0049] In a 250 ml glass bottle, 6.4 g of BSA, Miles Inc. was
dissolved in 80 ml of phosphate buffered saline (PBS) and 20 g of
10% Pt/XC72R carbon, MCA Ltd, was gradually added with constant
stirring. The bottle was then placed on a roller mixer and allowed
to incubate for two hours at room temperature.
[0050] A Buchner funnel was prepared with two pieces of filter
paper, Whatman.TM. No 1. The mixture was poured into the funnel and
the carbon washed three times with approximately 100 ml of PBS. The
vacuum was allowed to pull through the cake of carbon for about 5
minutes to extract as much liquid as possible. The cake of carbon
was carefully scraped out into a plastic container and broken up
with a spatula. The carbon was then placed in an oven at 30.degree.
C. overnight to dry. The purpose of this procedure is to block
active sites on the carbon hence to aid the shelf stability and
reproducibility of the carbon's properties.
Preparation of Platinum Group Metal/Carbon Inks
[0051] BSA-Pt/Carbon was prepared in Metech 8101 polyester resin as
the polymer binder and Butyl Cellosolve Acetate (BCA) as a solvent
for the ink. TABLE-US-00001 Ink Formulation Metech 8101 resin
44.68% BSA-Pt/Carbon 18.42% graphite 9.64% BCA/cyclohexanone 22.94%
Tween .RTM. 20 2.94% glucose oxidase 1.38%
[0052] Tween 20 is a surfactant supplied by Sigma-Aldrich. Tween is
a registered trade mark of ICI Americas, Inc. The solvent is a 50%
v/v mixture of BCA and cyclohexanone. The graphite was Timrex KS 15
(particle size <16 .mu.m), from GS Inorganics, Evesham, Worcs.
UK.
[0053] The resin, Tween 20, and about half the solvent were
initially blended together prior to adding the carbon fraction and
the graphite. Initially the formulation was hand-mixed followed by
several passes through a triple roll mill. The remaining volume of
solvent was then added to the ink and blended to bring the ink to a
suitable viscosity for printing.
Preparation of Drop-Coating Solutions
[0054] The coating solution is water-based and consists of a high
concentration of buffer, preferably phosphate at pH 8. It has been
found that buffering capacity is more important than ionic
strength. In this example the solution contains glucose oxidase and
a system stabiliser, in this example trehalose. TABLE-US-00002
Drop-Coat Solution Buffer KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 385 mM,
pH 8 Sigma Enzyme Glucose oxidase 4080 U/ml Biozyme Stabiliser
Trehalose 1% Sigma Preferred Ranges Buffer 300-1000 mM, pH 7-10
Enzyme 500-12000 U/ml (1.85-44.4 mg/ml) Stabiliser 0.5-30%
[0055] The activity of the glucose oxidase is about 270 units per
milligram of material (360 units/mg of protein because the enzyme
comes in a preparation with other lyophilisation and stabilisation
agents).
[0056] If the enzyme is located in the base layer the drop coating
solution may contain only buffer, optionally with the
stabiliser.
Methods of Manufacture
[0057] Glucose test strips (biosensors) were manufactured using a
combination of screen printing and drop coating technologies. Other
printing and/or coating technologies, well known per se to those
skilled in the printing and coating arts may also be used. The
exemplified methods are by way of illustration only. It will be
understood that in each case the order of performance of various
steps may be changed without affecting the end product. For each of
FIGS. 1-2 the top row illustrates a process step, and the bottom
row illustrates the sequential build-up of the biosensor.
[0058] To manufacture the biosensor shown in FIG. 1, a base
substrate 2 is formed from a polyester material (Valox.TM.).
Conductive tracks 4 were printed onto the substrate 2 as a
Conductive Carbon Paste, product code C80130D1, Gwent Electronic
Materials, UK. In this embodiment, tracks 4a, 4b and 4c provide
electrical contacts for connecting, respectively, the working
electrode 24, the counter electrode 23 and the reference electrode
22 to a meter (not shown). Conductive track 4a' is printed to
provide a conductive surface on which the working electrode 24 will
be formed. After printing, the ink of the conductive tracks 4 was
dried for 1 minute in a forced air dryer at 130.degree. C. The
second ink printed on top of the conductive carbon 4 is a
Silver/Silver Chloride Polymer Paste, product code C61003D7, Gwent
Electronic Materials, UK. In this example, track 6a connects the
conductive carbon tracks 4a and 4a'; tracks 6b and 6c are connected
to the respective conductive carbon tracks 4b and 4c, and will
provide, respectively, the counter electrode 23 and the reference
electrode 22. The ink 6 is dried at 130.degree. C. in a forced air
dryer for 1 minute.
[0059] The next layer is the platinum group metal carbon ink which
is printed onto the conductive carbon 4d where the working
electrode 24 is to be formed. This ink is dried for 1 minute at
90.degree. C. in a forced air dryer to form a conductive base layer
8a about 12 .mu.m thick. A dielectric layer 10 is then printed,
excluding a working area 12 in which the working 24 and reference
22 electrodes are to be located. The dielectric layer 10 is MV27,
from Apollo, UK. The purpose of this layer is to insulate the
system. It is dried at 90.degree. C. for 1 minute in a forced air
dryer. If desired, the base layer 8a can alternatively be printed
after the dielectric layer 10. However, it is preferred to print
the base layer 8a first, since the subsequent application of the
dielectric layer 10 removes some of the tolerance requirements of
the print.
[0060] A drop-coat layer is applied to the base layer 8a using
BioDot drop-coating apparatus. The volume of drop-coating solution
used is 125 nl, applied as a single droplet; the drop-coat layer is
dried in a forced air dryer for 1 minute at 50.degree. C. to form
the working electrode 24. After drop-coating, the
partially-constructed test strips were allowed to condition for
four days at room temperature and low humidity. A spacer layer 14
is formed by screen-printing a UV-curable resin (Nor-Cote 02-060
Halftone Base) on the dielectric layer 10 and then curing the resin
with UV light (120 W/cm medium pressure mercury vapour lamp) at up
to 30 m/min. The resin comprises acrylated oligomers (29-55%)
N-vinyl-2-pyrrolidone (5-27%) and acrylated monomers (6-28%). The
spacer 14 has a channel 16 which will determine the capillary flow
path of the biosensor. A second substrate, or lid, 18 is adhered to
the spacer 14 to produce the biosensor 20. The lid 18 comprises a
50 .mu.m PET tape (Adhesive Research 90119) coated with about 12.5
.mu.m of a hydrophilic heat-seal adhesive `HY9`. The lid 18 is
adhered to the spacer 14 by the action of heat and pressure
(100.degree. C., 400 kPa) for 1-2 seconds. The lid 18 is provided
with a narrow vent 19 to permit the exit of air from the capillary
flow path. The vent 19 need not extend right across the lid 18 but
could comprise a hole or short slot in fluid communication with the
capillary flow path. If desired, the second substrate 18 may be
laid over a number of substrates on which the above steps are
carried out, followed by guillotining of the lid 18 to produce the
biosensor 20. Alternatively the spacer 14 could be initially
adhered to the second substrate 18 and then adhered to the first
substrate. A benefit of this arrangement is that the second
substrate 18 may be cut to provide the vent 19 while both parts of
the second substrate 18 are held in the correct positions by the
spacer 14.
[0061] The biosensor 20 has a reference electrode 22, a counter
electrode 23 and a working electrode 24 which are defined by the
working area 12 in the dielectric layer 10. The working electrode
24 comprises the base layer 8a on a conductive carbon layer 4a' on
the first substrate 2, and a top layer including the buffer.
[0062] In large-scale manufacturing, a plurality of substrates may
be provided initially connected together on a single blank or web,
preferably two substrate-lengths deep, and the various processing
steps carried out on the entire blank or web, followed by a final
separation step to produce a plurality of biosensors 20.
[0063] The biosensor 20 has a capillary flow path defined by the
channel 16 in the spacer 14, the inner surface of the lid 18, and
the first substrate 2 (largely covered by the dielectric layer 10).
The flow path extends from an inlet 17 at the parallel short edges
of each of the substrates 2, 18 to the reference, counter and
working electrodes 22, 23, 24. The inner surface of the lid 18 is
treated to be hydrophilic to facilitate wetting by blood. With
glucose oxidase as the enzyme, the biosensor is used to measure
blood glucose. A user may take a reading by pricking an alternative
site such as his or her upper arm to produce a small drop of blood
on the skin, and touching the appropriate short edge of the
biosensor 20 to the skin where the blood is located. The blood is
drawn rapidly to the working area 12, producing a current readable
by a meter connected to the conductive tracks 4a, 4b, 4c in a known
manner. A sample volume of about 0.8 nl is sufficient. However, if
an insufficient sample volume is applied, an inaccurate reading may
result. Application of a second sample will then cause a
non-faradaic charging peak, as will be discussed later.
[0064] Referring now to FIG. 2, an alternative embodiment of
biosensor is shown. In this embodiment, the conductive carbon
tracks 4a, 4b, 4c extend to the working area 12, and Ag/AgCl tracks
are not used. Each track 4a, 4b, 4c is printed with a base layer
8a, 8b, 8c respectively, using the same formulation and printing
method as for the base layer 8a of FIG. 1. The further processing
steps are identical to those for FIG. 1, with the drop coat being
applied only to the base layer 8a which will form the working
electrode 24. However, for manufacturing convenience it would also
be possible to apply the top coat to all of the base layers 8a, 8b
and 8c, using any convenient coating or printing technique, thereby
removing the need for accurate positioning of a droplet on a
precise area 8a.
[0065] A meter 42 suitable for use with the biosensors 20 is shown
schematically in FIG. 5. The meter 42 has electrode contacts 26,
27, 28 for making contact, respectively, with the electrode tracks
4a, 4b, 4c on the biosensor 20. Electrical values are communicated
to a processor 32 via an A/D converter 30. The processor 32 can
selectively access first storage means 37 storing calibration data
relating to whole blood and, optionally, to second storage means 38
storing calibration data relating to a control solution. In this
example, the calibration data 37, 38 are contained on a code chip
36 which is provided with a pot of biosensors 20. The processor 32
has a temperature sensor 40, and calibrates output analyte values
using the measured temperature and calibration data 37 or 38. The
calculated analyte value is displayed on an LCD 34.
[0066] When an analyte measurement is to be made, a biosensor 20 is
inserted into the meter 42 so that the electrode tracks 4a, 4b, 4c
make electrical contact with the respective electrode contacts 26,
27, 28. Insertion of the biosensor 20 completes a circuit and
activates the meter 42 to expect a measurement within a specified
time, in this example, two minutes. To take a blood glucose
measurement, a user pricks a finger or alternative site such as the
upper arm, to provide a drop of blood, and touches the free end of
the biosensor 20 against the drop. Capillary forces draw the blood
sample into the biosensor via the channel 16 of the capillary flow
path. The arrangement of electrodes is such that the blood sample
makes contact with the working electrode 24 no later than it makes
contact with the reference electrode 22. When the sample makes
contact with both the reference 22 and working 24 electrodes this
completes a circuit and alerts the processor 32 that a sample has
made contact with the working electrode 24. The processor 32
includes first signal means 31 for producing a first signal when
this circuit is completed. The processor 32 also comprises a timer
35 which measures the time from when the first signal is
produced.
[0067] Further flow of the fluid sample down the channel 16 of the
capillary flow path brings the sample into contact with the
separate counter electrode 23. This completes an electric circuit
between the counter electrode 23 and/or the reference electrode 22
and the working electrode 24, and alerts the processor 32 that the
sample has wholly or sufficiently covered the working electrode 24
for a reading to be taken. The processor 32 includes second signal
means 33 for producing a second signal when this second circuit is
completed. If the second signal is not produced within a pre-set
time, for example five seconds, after the first signal, the
processor will trigger an error condition which will be displayed
on the LCD 34. The user will be prompted to remove the used
biosensor 2 and begin again with a fresh biosensor. If insufficient
sample is present to produce the second signal, the user may be
able to augment the sample by applying a second drop of blood. This
double-dosing does not generate a non-faradaic charging peak
because the circuit has not yet been completed between the working
and counter electrodes. When the second signal has been produced
within the pre-set time, the working electrode is polarised, for
example to 350 mV or 375 mV, and an electrochemical measurement, in
this example an amperometric measurement, is made.
[0068] It will be understood that the first signal means 31 and the
second signal means 33 may be provided by one and the same
circuitry, and that the processor 32, the signal means 31, 33,
timer 35, and storage means 37, 38 may each be provided as separate
components or that any or all of them may be provided in
combination, for example in a single processing unit.
[0069] Referring now to FIG. 6, a potentiostat circuit for a meter
in accordance with a preferred embodiment is illustrated. The
circuit has a first op-amp (43) and a second op-amp (44). 43 sets
the polarising potential at the working electrode (WE) relative to
the reference electrode (RE). At the start of a test, a switch 45
is made between contacts 27 and 28, thereby establishing a normal
combined reference/counter electrode configuration. When blood is
applied and establishes an electrical connection between the
reference and working electrodes, a current will flow. This current
is detected and the switch 45 is released. After the switch is
released no current will flow because the reference electrode will
be open circuit and the output of 44 will be at the same voltage as
the working electrode. When the blood sample reaches the reference
electrode, a feedback loop is made and normal operation of the
potentiostat circuit is resumed. When this happens the strip has
been filled with enough blood.
[0070] Test results for the biosensor of FIG. 1 are shown in FIG.
3, and test results for the biosensor of FIG. 2 are shown in FIG.
4. In both cases, the drop-coat solution was applied by
drop-coating onto the base layer 8a to form the working electrode
24, but not onto the reference electrode 22 or counter electrode
23. However, it would be possible to apply the drop-coat solution
to either or both of the other electrodes, or over the entire
working area 12 if convenient to do so. Both biosensors give an
acceptable response curve of current -v- glucose concentration, but
the biosensor of FIG. 2, in which all three electrodes comprise the
base layer 8 of platinised carbon, gives a higher background
current and is more sensitive and has a more linear response.
[0071] It is appreciated that certain features of the invention,
which are for clarity described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention which
are, for the sake of brevity, described in the context of a single
embodiment, may also be provided separately or in any suitable
subcombination.
[0072] While the present invention has been described with
reference to specific embodiments, it should be understood that
modifications and variations of the invention may be constructed
without departing from the spirit and scope of the invention
defined in the following claims.
* * * * *